Alpha1‐adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: Evidence for compartmentalization of quin‐2 and fura‐2

Abstract

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca²⁺]i) during the response to α₁-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca²⁺]_i. The basal level of [Ca²⁺]_i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca²⁺]_i. Phenylephrine (10⁻⁵ M) caused an immediate actual increase in [Ca²⁺]_i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca²⁺]_i averaging 22% and 44% over the basal level (180–300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca²⁺-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells “scrape-loaded” with fura-2 revealed a four-fold increase in [Ca²⁺]_i concentration following stimulation with phenylephrine.